Journal

of Hospital

Infection

(1992) 22, 163-178

Letters

to the Editor

Sir, Diversity

of klebsiellae with extended-spectrum Turkish University Hospital

p-lactamases

at a

‘Extended-spectrum’ p-lactamases differ in sequence by only one to four amino-acid residues from their parent TEM-1, TEM-2 or SHV-1 enzymes, but hydrolyse a wider range of antibiotics, typically including all cephalosporins except cephamycins.’ In most of the many hospitals where they have caused resistance problems these enzymes have been observed first in klebsiellae, but often have later spread to other enterobacteria.’ This predilection for klebsiellae remains unexplained. Their parent enzymes, particularly TEM-1, are widespread in other species. A possible explanation lies in the fact that klebsiellae survive longer than other enterobacteria on hands and surfaces, facilitating cross-infection.* Thus, a single strain, advantaged by its P-lactamase, might spread amongst patients. This process was apparent, for example, with a serotype K25 SHV-4 P-lactamase-producing strain in Paris.3 Hacettepe University Hospital, with 800 beds, is one of the largest teaching hospitals in Turkey. New generation cephalosporins, principally ceftazidime, cefotaxime and ceftriaxone, have been used extensively since the mid-1980s. Klebsiellae with relative resistance (minimum inhibitory concentration (MIC) 22 mg l-‘, compared to MICs 60.25mg 1-l ceftazidime or do.1 mg 1-l of cefotaxime and ceftriaxone for normal isolates) to one or more of these drugs were first observed in 198S4 and now account for about 25% of all K. pneumonia from inpatients.5 We investigated the characteristics of a random sample of 17 cephalosporin-resistant K. pneumoniae recovered from patients at the hospital during 1989-90. Their identity was confirmed by the API20E system (Bio-Merieux, La Balme les Grottes, France). Serotypes were determined by counter-current immunoelectrophoresis6 and by the Quellung reaction. ’ MICs of antibiotics were determined on DST agar (Oxoid CM261) plates with inocula of lo4 colony forming units (cfu) taken from overnight nutrient broth cultures. Crude J3-lactamase extracts were prepared by sonication of late exponential phase cells. Isoelectric focusing was performed essentially by the method of Matthew et aZ.,* but at a constant power of 10 W. The focusing gels were developed with 1 mM nitrocefin. Plasmid transfers were attempted by a plate mating method,’ crossing exponential phase cultures of the klebsiellae with Escherichia coli K-12 J53-1 (pro nul). Transconjugants were selected on DST agar containing ceftazidime (1 or 10 mg 1-l) or ampicillin (250 mg 1-l) plus nalidixic acid (100 mg 1-l). Plasmid extraction and electrophoresis was 0195-6701/92/100163+,6

tOS.OO/O

0 1992 The Hospital

163

Infectmn

Society

164

Letters

to the Editor

undertaken by the method of Kado and Liu.” The SHV specific probe and the hybridization method for reaction of this probe with total DNA cell extracts were as described by Liu et al.” The hydrolytic activity of p-lactamases was measured against 0.5 mM antibiotic solutions by UV spectrophotometry at 37°C in 10 mM phosphate buffer. The assay wavelengths and cuvette lightpaths were: ampicillin, benzylpenicillin and carbenicillin 235 nm, 1 cm; cephaloridine 295 nm, 1 cm; cefotaxime and ceftazidime 257 nm, 1 mm. Table I shows MIC data, together with details of serotype and J3-lactamases for the isolates. Brief details of the plasmid bands detected are also given. Although the cephalosporin MICs varied considerably amongst the isolates, all the organisms except no. 11544 were relatively resistant to one or more of cefotaxime, ceftriaxone or ceftazidime (MIC 2 2 mg 1-l) and showed an eight-fold or greater increase in susceptibility when the cephalosporin was combined with 4 mg 1-’ clavulanate. Strain 11544 was susceptible to 0.25 mg 1-l of cefotaxime, ceftriaxone and ceftazidime but still showed eight-fold greater susceptibility to the first two agents when these were combined with clavulanate 4mg 1-l. Susceptibility to cefoxitin or imipenem was, at most, only slightly reduced compared to typical klebsiellae, with MICs never exceeding 16 and 1 mg 1-i respectively. This is typical of production of extended-spectrum resistance pattern focusing revealed one to three P-lactamases in P-1actamases.’ Isoelectric each isolate. These variously co-focused with TEM-1 (p1 5*4), SHV-1 (p1 7*6), or had p1 values of 7.0, 8.2 or 8.4. Eleven different capsular serotypes were noted amongst the isolates, with no more than four isolates belonging to any one serotype. Multiple plasmids were detected in many organisms and no single plasmid type was consistently found amongst the isolates. Three isolates (11507, A4, 11595) appeared identical, as regards serotype (60), J3-lactamase profile (enzymes of p1 5.4, 7.6 and 8*2), plasmid profile (five identical bands) and antibiogram. One further isolate (11394) resembled these, but had only four of six plasmid bands co-incident on electrophoresis, was less resistant and had a p1 8.4. instead of 8.2, p-lactamase. Two further isolates (5718,472l) were identical to one another as regards serotype (54) J3-lactamase pIs (5*4,7*6) and plasmid profile (three identical bands), but differed in their degree of cephalosporin resistance. Otherwise, all the isolates were clearly distinct. Ampicillin resistance was transferred to E. coli 553-l by many of the organisms and was associated with p1 5.4 (TEM-1 co-focusing) J3-lactamases. Cross-resistance to third generation cephalosporins was never conferred by acquisition of such an enzyme in isolation. Resistance to third generation cephalosporins was transferred in five cases only and was associated with acquisition of the transconjugants of p1 7.6 p-lactamases either alone or in combination with other (p1 5.4 or 8.4) enzymes (Table I). Hydrolysis assays were performed with extracts prepared from cephalosporin-resistant transconjugants that acquired only p1 7.6 enzymes. Results are shown in Table II. The enzymes were similar in their hydrolytic

* P-Lactamases

Clav = clavulanic

1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 16 > 1024 > 1024 > 1024

> > > > > > > > > > > >

> 1024

that were transferred

acid 4 mg IV’. NT=

2 NT

10

66: 54 54 43 35 19 ;:

60

79

79 2:

5441 Bah 11507 A4 11595 11394 5718 4721 365 K20 240 Ferdi 11544 5561 7184 34956

Serotype

37

Isolate

Ampicillin

>I024 8 8 8 128 256 2.56 256 128 64 32 32 8 16 2 32 32 32

to E. coli recipients,

not typable.

>I024 >I024 >I024 >I024 >1024 > 1024 >I024 >I024 >I024 >I024 >I024 >I024 16 >I024 >I024 >I024

2

giving

8 16 >I28 >I28 >I28 64 2 8 2 2 8 64 0.25 4 128 2

2

0.03

generation

0.12 0.03 0.12 0.12 0.12 0.12 0.03 0.03 ,I28 >I28 >I28 16 2 16 2 :

Alone

Cefotaxime

(mg 1-l)

of Klebsiella

resistance

0.25 0.5 2 1 1 1 0.5 0.5 0.5 0.25 0.5 0.25 0.25 0.25 0.25 0.25

0.25

+ Clav

Alone

Alone

+ Clav

Ceftazidime

MIC

I. Characteristics

Amoxycillin

Table

;:: 0.25

1 0.25 0.25 0.5 0.5 0.5 0.5 0.5

0.5 0.25 1 :

0.5

Imipenem

:

;

i?J 4 4 :.

5

:

No. of plasmid bands

5.4, 7.6 7.6* 5.4, 7.6, 8.2 5.4, 7.6, 8.2 5.4, 7.6, 8.2 5.4, 7.6, 8.4 5.4, 7.6 5.4, 7.6* 5.4, 7.0, 7.6 5.4, 7.6 7.6* 7.6, 8.2 7.6* 5.4, 7.6 5.4, 7.6, 8.4 5.4, 7.6

7.6

PIS

p-lactamase

g 5 1

r =: 2 z 0 g

166

Letters

Table

I I. Substrate projiles of pI 76J-lactamase

extracts of transconjugants preparedfrom four klebsiella strains

Relative Donor Bah 4721 11544 240 Control

to the Editor

strain

Penicillin

SHV-1

100 100 100 100 100

Ampicillin 312 479 365 283 150

rate of hydrolysis Carbenicillin 29 31 45 28 6.0

ZIS. 0.5 mM antibiotic Cefotaxime 67 68 50 51

Diversity of klebsiellae with extended-spectrum beta-lactamases at a Turkish university hospital.

Journal of Hospital Infection (1992) 22, 163-178 Letters to the Editor Sir, Diversity of klebsiellae with extended-spectrum Turkish University...
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